DNA-binding proteins play critical roles in regulating a wide range of cellular processes important for normal development and growth. The Cys2His2 zinc finger (hereafter "zinc finger") is the most common DNA-binding motif encoded in genomes ranging from yeast to human. Zinc fingers typically occur in tandem arrays in transcription factors and these proteins bind specifically to a wide variety of target DNA sequences. The zinc finger motif has also emerged as the scaffold of choice for creating customized DNA-binding domains targeted to any DNA sequence of interest. Although these "designer" zinc finger domains have shown tremendous potential for applications in biological research and gene therapy, robust methods for consistently producing proteins with single-gene specificity in human cells have yet to be described. [unreadable] [unreadable] Certain naturally occurring zinc finger proteins bind to extended DNA sequences using arrays consisting of six or more zinc fingers but little is understood about how these proteins recognize their cognate DNA sites. We propose to perform genetic, biochemical and structural characterization of the neuron restrictive silencer factor (NRSF, also known as the RE-1 silencing transcription factor or REST), a biologically important transcriptional repressor that uses eight zinc fingers to bind its cognate 21 base pair DNA sequence. Additionally, we will test whether NRSF may provide a robust scaffold for creating designer zinc finger proteins with single-gene targeting specificities in human cells. These studies will substantially improve our knowledge of NRSF function, with broad biological significance for understanding other extended zinc finger-DNA interfaces. In addition, our studies will facilitate efforts to create highly specific synthetic DNA-binding domains capable of recognizing unique addresses within mammalian genomes. [unreadable] [unreadable]